Small molecules play an important role in many biological functions. Because small molecules can alter the functions of proteins, for example, by binding to them and inhibiting or activating their normal functions, they can perturb/disrupt the systems in which the protein operates. In turn, such disruptions can trigger diseases or accelerate the progression of diseases. By controlling small molecule dosage, on the other hand, one can uncover the details about the role(s) the protein plays within these systems (Stockwell, Brent R. “Exploring biology with small organic molecules.” Nature 432.7019 (2004): 846-854). Detecting the presence and concentration of small molecules can also be used to assess the result of a metabolic process, such as drug metabolism, which could benefit and accelerate clinical trials and drug efficacy studies. Beyond human and animal diagnostics, small molecules can also be used to assess the status of the environment, including for monitoring agricultural plant stress and water quality. The ability to detect and accurately quantitate small molecules is therefore valuable across many domains of biotechnology.
There are a number of available methods to measure small molecules; some are inexpensive, but lack accuracy and sensitivity, others are very sensitive, but costly and complex. Less expensive high throughput methods generally involve binding a dye to the target molecule of interest and detecting aggregate fluorescence or absorbance of the sample. This technique can be hampered by non-specificity of the dye or interfering molecules that create high false positives and/or high false negatives. More complex analytical methods, including high performance liquid chromatography and mass spectrometry, are able to precisely measure target small molecules, but are complex and costly and require dedicated lab space and trained personnel. What is needed, therefore, is a method of detecting small molecules that is cost effective (similar to a dye binding assays), provides high specificity, sensitivity, and accuracy, and can be performed on a portable device. A preferred analytical test for detection and/or quantification of small molecules would not be tethered to a dedicated lab and would not require specialized equipment or trained personnel. Additionally, a preferred analytical device would be of low cost and high reproducibility. Therefore, methods for providing accurate and low-cost analytical results on low cost devices that tolerate a range of nanopore geometries and/or larger nanopores are needed.